From New DNA Conjugation Approaches to 3D DNA Networks for an Artificial Extracellular Matrix
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The aim of this work was to establish a material based on branched DNA, with cell attractive properties. Several challenges had to be addressed to reach this goal. First of all, the building blocks for automated DNA synthesis had to be efficiently synthesized. Two synthesis routes were elaborated to synthesize bis homotris-based branched DNA building blocks with exchangeable chemical handles at the branching core. Three distinct synthesis strategies at the automated DNA synthesizer were tested for applicability. As inverse DNA synthesis is unavoidably needed, two strategies aimed at only synthesizing one DNA strand with this less efficient DNA synthesis, to raise the overall yield of branched DNA. These strategies relied on the bridging of two synthesized DNA strands on the solid support. To achieve efficient bridging, only low excesses of the branched-DNA building block could be applied, resulting in only low coupling efficiencies. As these strategies appeared to be inefficient, a synthesis strategy was optimized that relied on the parallel synthesis of two DNA strands with inverse DNA synthesis. To ensure the best possible yields, the general synthesizer chemistry was optimized. Thereby, the exchange of the activating agent ethylthio-1H-tetrazole with 4,5-dicyanoimidazole increased the yields significantly, providing the foundation for the efficient synthesis of branched DNA constructs, bearing free 3’-ends as needed for elongation by DNA polymerases. The enzymatic elongation of the branched primers was optimized. Especially with the surface-bound application in mind, the concentration of additives was optimized, to ensure efficient PCR with the branched primers. Additives like BSA were needed, to passivate surfaces that can come in contact to the DNA. Efficient PCR protocols were elaborated. To form DNA networks by branched PCR on glass slides, DNA had to be immobilized an glass substrates. The spotting procedures had to be optimized, particularly in respect to unselective, electrostatic immobilization of DNA. Amino functionalized glass slides were activated by p phenylenediisothiocyanate treatment. Unreacted amino groups had to be passivated by a treatment of succinic anhydride and N-methylimidazole, to convert the positive charge of the amino function to a negative charge of the succinic acid. DNA spotting protocols and subsequent passivation steps were optimized, so branched DNA modified glass slides are now reliably obtainable. Considering the modification of the branched DNA networks, bioorthogonal ligation reactions were tested towards their applicability. As promising reactions, the copper catalyzed alkyne-azide cycloaddition and the Diels-Alder reaction were tested. As the building block for the synthesis of branched DNA already had a pentynoic acid-moiety, the copper catalyzed reaction was explored first. Different Cu+-stabilizing ligands were synthesized and tested. BTTAA showed by far the best properties to be used with the branched DNA constructs. Protocols were elaborated for the efficient ligation of branched primers. Additionally, the possibility for the modification of DNA strands by the incorporation of alkyne-bearing dNTPs was explored. Two dNTPs were explored, that were alkyne modified and were readily incorporated by enzymatic synthesis. The ligation protocols elaborated for the branched DNA worked efficiently on the intra-strand modified DNA. The approach for incorporating modified building blocks by enzymatic reaction and post-synthetic labelling of the DNA was transferred to modification by Diels-Alder reaction. For that reason, vinylated dNTPs were synthesized and their incorporation into DNA by primer extension experiments was tested. Both dvinATP and dvinUTP were incorporated efficiently by KlenTaq DNA polymerase, but upon incubation with a biotin-modified tetrazine, only the dvinAMP containing DNA underwent inverse electron demand Diels-Alder reaction. Measurements of the reaction kinetics revealed even higher conversion rates on DNA as on the isolated nucleosides. All in all, these favorable reaction kinetics prompted the inverse electron demand Diels-Alder reaction a very promising labelling reaction. With labelling reactions in hand, suitable bio-active molecules, so called bait molecules, were tested for their attachment to the DNA networks. These bioactive compounds should lead to cell attachment to the DNA networks. As bait molecule a cyclic peptide was established. This peptide presenting the well-known RGD motif can serve as attachment site for various cell types, as the RGD-motif is the universal recognition sequence in many multicellular organisms. An azide-functionalized version was obtained. The modification of surface-bound DNA by CuAAC was tested. A fluorescent azide was used in first studies, to detect efficient conversion of the DNA networks on the slides by reading the fluorescence signal. No efficient ligation protocols could be established directly on the glass slides. But the conversion of branched primers with either a biotin azide or the azido-functionalized cRGD-peptide was possible. So, modified branched primers were obtained, which were used for the generation of surface bound bioactive DNA networks. With these networks, first proof of concept studies were performed. HeLa cells were cultured on DNA modified slides, with and without bait molecules. It was shown, that to the bait molecule-modified DNA networks double as many cells adhered as to the unmodified DNA networks. After stressing of the cells and prolonged incubation, up to 7-fold more cells adhered to the functionalized networks compared to unmodified DNA networks. To conclude, a cell attractive material could be generated and the first experiments towards its application are very promising.
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BUSSKAMP, Holger, 2015. From New DNA Conjugation Approaches to 3D DNA Networks for an Artificial Extracellular Matrix [Dissertation]. Konstanz: University of KonstanzBibTex
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title={From New DNA Conjugation Approaches to 3D DNA Networks for an Artificial Extracellular Matrix},
author={Bußkamp, Holger},
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<dcterms:abstract>The aim of this work was to establish a material based on branched DNA, with cell attractive properties.
Several challenges had to be addressed to reach this goal. First of all, the building blocks for automated DNA synthesis had to be efficiently synthesized. Two synthesis routes were elaborated to synthesize bis homotris-based branched DNA building blocks with exchangeable chemical handles at the branching core.
Three distinct synthesis strategies at the automated DNA synthesizer were tested for applicability. As inverse DNA synthesis is unavoidably needed, two strategies aimed at only synthesizing one DNA strand with this less efficient DNA synthesis, to raise the overall yield of branched DNA. These strategies relied on the bridging of two synthesized DNA strands on the solid support. To achieve efficient bridging, only low excesses of the branched-DNA building block could be applied, resulting in only low coupling efficiencies. As these strategies appeared to be inefficient, a synthesis strategy was optimized that relied on the parallel synthesis of two DNA strands with inverse DNA synthesis. To ensure the best possible yields, the general synthesizer chemistry was optimized. Thereby, the exchange of the activating agent ethylthio-1H-tetrazole with 4,5-dicyanoimidazole increased the yields significantly, providing the foundation for the efficient synthesis of branched DNA constructs, bearing free 3’-ends as needed for elongation by DNA polymerases.
The enzymatic elongation of the branched primers was optimized. Especially with the surface-bound application in mind, the concentration of additives was optimized, to ensure efficient PCR with the branched primers. Additives like BSA were needed, to passivate surfaces that can come in contact to the DNA. Efficient PCR protocols were elaborated.
To form DNA networks by branched PCR on glass slides, DNA had to be immobilized an glass substrates. The spotting procedures had to be optimized, particularly in respect to unselective, electrostatic immobilization of DNA. Amino functionalized glass slides were activated by p phenylenediisothiocyanate treatment. Unreacted amino groups had to be passivated by a treatment of succinic anhydride and N-methylimidazole, to convert the positive charge of the amino function to a negative charge of the succinic acid. DNA spotting protocols and subsequent passivation steps were optimized, so branched DNA modified glass slides are now reliably obtainable.
Considering the modification of the branched DNA networks, bioorthogonal ligation reactions were tested towards their applicability. As promising reactions, the copper catalyzed alkyne-azide cycloaddition and the Diels-Alder reaction were tested. As the building block for the synthesis of branched DNA already had a pentynoic acid-moiety, the copper catalyzed reaction was explored first. Different Cu+-stabilizing ligands were synthesized and tested. BTTAA showed by far the best properties to be used with the branched DNA constructs. Protocols were elaborated for the efficient ligation of branched primers. Additionally, the possibility for the modification of DNA strands by the incorporation of alkyne-bearing dNTPs was explored. Two dNTPs were explored, that were alkyne modified and were readily incorporated by enzymatic synthesis. The ligation protocols elaborated for the branched DNA worked efficiently on the intra-strand modified DNA.
The approach for incorporating modified building blocks by enzymatic reaction and post-synthetic labelling of the DNA was transferred to modification by Diels-Alder reaction. For that reason, vinylated dNTPs were synthesized and their incorporation into DNA by primer extension experiments was tested. Both dvinATP and dvinUTP were incorporated efficiently by KlenTaq DNA polymerase, but upon incubation with a biotin-modified tetrazine, only the dvinAMP containing DNA underwent inverse electron demand Diels-Alder reaction. Measurements of the reaction kinetics revealed even higher conversion rates on DNA as on the isolated nucleosides. All in all, these favorable reaction kinetics prompted the inverse electron demand Diels-Alder reaction a very promising labelling reaction.
With labelling reactions in hand, suitable bio-active molecules, so called bait molecules, were tested for their attachment to the DNA networks. These bioactive compounds should lead to cell attachment to the DNA networks. As bait molecule a cyclic peptide was established. This peptide presenting the well-known RGD motif can serve as attachment site for various cell types, as the RGD-motif is the universal recognition sequence in many multicellular organisms. An azide-functionalized version was obtained.
The modification of surface-bound DNA by CuAAC was tested. A fluorescent azide was used in first studies, to detect efficient conversion of the DNA networks on the slides by reading the fluorescence signal. No efficient ligation protocols could be established directly on the glass slides. But the conversion of branched primers with either a biotin azide or the azido-functionalized cRGD-peptide was possible. So, modified branched primers were obtained, which were used for the generation of surface bound bioactive DNA networks.
With these networks, first proof of concept studies were performed. HeLa cells were cultured on DNA modified slides, with and without bait molecules. It was shown, that to the bait molecule-modified DNA networks double as many cells adhered as to the unmodified DNA networks. After stressing of the cells and prolonged incubation, up to 7-fold more cells adhered to the functionalized networks compared to unmodified DNA networks.
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